Ribose QC Manual

User Manual:

Open the PDF directly: View PDF .
Page Count: 34

Download
Open PDF In Browser	View PDF

Package ‘RiboseQC’
May 31, 2019
Title Ribo-seQC, a comprehensive Ribo-seq analysis tool
Version 0.99.0
Description Ribo-seQC is a powerful analysis tool for the analysis of Riboseq data, which is able to provide read-length specific analysis of both cytoplasmic and organellar ribosome, and provides interactive visualization of results in a dynamic html report
Depends rmarkdown, rtracklayer, GenomicAlignments, BSgenome,
GenomicFiles, devtools, reshape2, ggplot2, knitr, DT,
gridExtra, ggpubr, viridis, Biostrings, GenomicFeatures,
BiocGenerics
License GPL-3 or above
Encoding UTF-8
LazyData FALSE
Name RiboseQC
biocViews RiboSeq, GenomeAnnotation, Transcriptomics, Software
RoxygenNote 6.1.1
NeedsCompilation no
Author Lorenzo Calviello [aut],
Dominique Sydow [aut],
Dermott Harnett [ctb, cre],
Uwe Ohler [rev, fnd]
Maintainer Dermott Harnett 

R topics documented:
calc_cutoffs_from_profiles . .
choose_readlengths . . . . . .
create_html_report . . . . . .
create_pdfs_from_rds_objects
generate_rdata_list . . . . . .
get_codon_usage_data . . . .
get_default_rl_selection . . . .
get_metagene_data . . . . . .

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
1

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.

2
3
4
5
6
7
8
9

2

calc_cutoffs_from_profiles
get_ps_fromsplicemin . . . . . . .
get_ps_fromspliceplus . . . . . .
get_rl_and_cutoffs . . . . . . . .
get_top50_all_genes . . . . . . .
get_top50_cds_genes . . . . . . .
get_top50_mapping . . . . . . . .
load_annotation . . . . . . . . . .
plot_codon_usage_bulk . . . . . .
plot_codon_usage_bulk_rmd . . .
plot_codon_usage_positional . . .
plot_codon_usage_positional_rmd
plot_frame_dist_boxplot . . . . .
plot_frame_dist_boxplot_rmd . .
plot_metagene_bar . . . . . . . .
plot_metagene_bar_rmd . . . . .
plot_metagene_hm . . . . . . . .
plot_metagene_hm_rmd . . . . .
plot_read_biotype_dist_1 . . . . .
plot_read_biotype_dist_2 . . . . .
plot_read_biotype_dist_by_length
plot_read_length_dist . . . . . . .
plot_read_length_dist_by_biotype
prepare_annotation_files . . . . .
RiboseQC_analysis . . . . . . . .

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

Index

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.

11
11
12
12
13
14
14
15
16
17
18
19
20
21
22
22
23
24
25
26
27
28
29
31
34

calc_cutoffs_from_profiles
Calculate offsets from 5’end profiles

Description
This function calculates cutoffs and frame resolution for Ribo-seq reads, for each read length and
compartment.
Usage
calc_cutoffs_from_profiles(reads_profile, length_max)
Arguments
reads_profile

Profile of 5’ends around start and stop codon, as a DataFrame object with tx_ids
as rows and positions as columns

length_max

Maximum cutoff to use

choose_readlengths

3

Details
Three methods are used and combined in the final choice: the position of maximum coverage
around start codon is calculated for each transcript, and the most frequent one is stored in the
"*_tab" objects. Such frequency values are also subjected to k-means clustering (centers=3) and
the first value belonging to the highest cluster is selected, output as "*km_tab" objects. Analysis
of aggregate plots, instead of frequencies, is performed again using kmeans (centers=3) using the
same analysis above and stored in the "*km_meta" objects, and by simply calculating the maximum
value in the profile, stored in the "*meta" objects. for each method, all reads ("absolute_") or only
in-frame positions ("in_frame_") are considered. The final choice takes the most frequent cutoff
chosen in all methods applied to in-frame positions.
Value
a list with a final_cutoff object, the frame analysis containing the displaying the max frame and
the average all the calculated cutoffs in cutoffs, data used for the frame analysis in frames, and
profiles around start codons in profiles_start.
Author(s)
Lorenzo Calviello, 
See Also
RiboseQC_analysis
choose_readlengths

Filter read lengths for P-sites position calculation

Description
This function selects a subset of readlenghts to be used in the P-sites calculation step
Usage
choose_readlengths(summary_data, choice = "max_coverage", nt_signals)
Arguments
summary_data
choice
nt_signals

output data from the calc_cutoffs_from_profiles function
Method used to select readlengths, defaults to "max_coverage"
Profiles of 5’ends around start codons

Details
Three different methods are available to choose readlengths: the "max_coverage" method selects
all read lenghts with more in-frame signal compared to out-of-frame signal, on all codons; the
"max_inframe" method starts with the most accurate read length and progressively selects read
lengths which add in-frame signals in codons not covered by previous read lengths; the "all" method
selects all available read lengths

4

create_html_report

Value
a list object containing different compartments. Each sub-list contains final_choice, the set of
chosen read lengths with cutoffs, and data, the complete stats for each selection method
Author(s)
Lorenzo Calviello, 
See Also
calc_cutoffs_from_profiles

create_html_report

Create the Ribo-seQC analysis report in html

Description
This function creates the Ribo-seQC html report based on the Ribo-seQC analysis files generated
with RiboseQC_analysis.
Usage
create_html_report(input_files, input_sample_names, output_file,
extended = F)
Arguments
input_files

Character vector with full paths to data files generated with RiboseQC_analysis.
Must be of same length as input_sample_names.
input_sample_names
Character vector containing input names (max. 5 characters per name). Must be
of same length as input_files.
output_file

String; full path to html report file.

extended

creates a large html report including codon occupancy for each read length. Defaults to FALSE

Details
This function creates the html report visualizing the RiboseQC analysis data.
Input are two lists of the same length:
a) input_files: list of full paths to one or multiple input files (Ribo-seQC analysis files generated with RiboseQC_analysis) and
b) input_sample_names: list of corresponding names describing the file content in max. 5 characters (these are used as names in the report).

create_pdfs_from_rds_objects

5

For the report, a RMarkdown file is rendered as html document, saved as output_file.
Additionally, all figures in the report are saved as PDF figures in an extra folder in the same directory as the report html file.
Example:
output_file <- "\mydir\myreport.html" will generate the html report \mydir\myreport.html
and the folder \mydir\myreport_plots\ for the RDS object files to be stored in.
Value
The function saves the html report file with the file path output_file and a folder containing all
figures shown in the html report as RDS object files (located in the same directory as the html
report).
Author(s)
Dominique Sydow, 
See Also

plot_read_biotype_dist_1, plot_read_biotype_dist_2, plot_read_length_dist, plot_read_length_dist_by_bio
plot_read_biotype_dist_by_length, get_metagene_data, plot_metagene_hm_rmd, plot_metagene_hm,
plot_metagene_bar_rmd, plot_metagene_bar, plot_frame_dist_boxplot_rmd, plot_frame_dist_boxplot,
get_rl_and_cutoffs, get_default_rl_selection, get_top50_mapping, get_top50_cds_genes,
get_top50_all_genes, get_codon_usage_data, plot_codon_usage_positional_rmd, plot_codon_usage_positional
plot_codon_usage_bulk_rmd, plot_codon_usage_bulk

create_pdfs_from_rds_objects
Generate PDF files from RDS object files

Description
This function generates figures as PDF files from RDS object files.
Usage
create_pdfs_from_rds_objects(output_rds_path)
Arguments
output_rds_path
String; full path to output folder for RDS object files. Example: /my_path_to/rds/
Value
This function creates PDF files from RDS object files.

6

generate_rdata_list

Author(s)
Dominique Sydow, 
See Also
create_html_report

generate_rdata_list

Generate a list of R data objects

Description
This function generates a list of loaded RData files to be used during Ribo-seQC report generation.

Usage
generate_rdata_list(input_files)
Arguments
input_files

List of RData file paths generated by RiboseQC_analysis.
Example:
input_files <- c(sample1="//path//to//sample1", sample2="//path//to//sample2")

Value
This function returns a list of loaded RData objects that were generated by RiboseQC_analysis.
Author(s)
Dominique Sydow, 
See Also
create_html_report

get_codon_usage_data

7

get_codon_usage_data

Get codon usage data (positional and bulk)

Description
This function processes codon usage data generated by RiboseQC_analysis, i.e. codon usage
• per nucleotide position (i.e. positional codon usage) as well as
• summed up over all positions* (i.e. bulk codon usage)
for a specific data type, originating compartment, and read length, as well as based on a user-defined
genetic code.
This data is used as input in plot_codon_usage_bulk to generate a bar plot, and in plot_codon_usage_bulk_rmd
to iterativly generate plots for the Ribo-seQC report in section 7.2.
Please check plot_codon_usage for positional codon usage (instead of bulk codon usage).
* Information on positions included in analysis:
Based on P-site corrected reads, the codon usage within CDS regions for protein coding genes is
examplatory calculated
• for the first 11 codons of the CDS (referred to as start),
• for 11 codons from the middle of the CDS (referred to as middle), and
• for the last 11 codons of the CDS (referred to as stop).
Usage
get_codon_usage_data(data, data_type, comp, rl)
Arguments
data

Object (list of lists) generated by RiboseQC_analysis: res_all.

data_type

String; select one of the following:
• Codon_counts: codon count in defined positions*,
• P_sites_percodon: P-sites count in defined positions*, or
• P_sites_percodon_ratio: ratio of P-sites counts to codon counts in defined positions*.
• E_sites_percodon: E-sites count in defined positions*, or
• E_sites_percodon_ratio: ratio of E-sites counts to codon counts in defined positions*.

8

get_default_rl_selection
• A_sites_percodon: A-sites count in defined positions*, or
• A_sites_percodon_ratio: ratio of A-sites counts to codon counts in defined positions*.
comp

String for originating compartment.
Check for available originating compartments in the data set using:
names(res_all$profiles_P_sites$Codon_count)

rl

String for read length.
Check for available read lengths in the data set using:
names(res_all$profiles_P_sites[[data_type]][[comp]])

Value
This function returns a list (e.g. called codon_usage_bulk_data) with information on bulk codon
usage:
• codon_usage_bulk_data$data contains the data on bulk codon usage,
• codon_usage_bulk_data$data_type saves the data type used (see parameter data_type)
• codon_usage_bulk_data$comp saves the originating compartment used (see parameter comp),
and
• codon_usage_bulk_data$rl saves the read length used (see parameter rl).
Author(s)
Dominique Sydow, 
See Also
create_html_report

get_default_rl_selection
Get default choice of read lengths

Description
This function returns selected read lengths per originating compartment. These read lengths build
the basis for all P-site based calculations such as metagene and codon usage analyses (e.g. plot_metagene_hm
and plot_codon_usage)
This data is used in the Ribo-seQC report in section 4.2.3 (displayed as table).
Usage
get_default_rl_selection(rdata_list)

get_metagene_data

9

Arguments
rdata_list

List of RiboseQC analysis RData objects generated by generate_rdata_list.

Value
This function returns data.
Author(s)
Dominique Sydow, 
See Also
create_html_report

get_metagene_data

Get 5’/P-site profile data for metagene analysis

Description
This function processes profile data generated by RiboseQC_analysis.
This data is used as input in plot_metagene_hm to generate plot for the Ribo-seQC report in section
4.1/4.3.
Usage
get_metagene_data(data, profile_type, res, comp)
Arguments
res

Resolution (subcodon or bins)
Subcodon resolution:
five_prime_subcodon
(in order to call res_all$profiles_fivepr$five_prime_subcodon) or
P_sites_subcodon
(in order to call res_all$profiles_P_sites$P_sites_subcodon)
Read coverage for the first 25nt after the transcription start site (TSS), 25nt
before and 33nt after the start codon, 33nt from the middle of the CDS, 33nt
before and 25nt after the stop codon, and the last 25nt before the transcription
end site (TES).
Bins:
five_prime_bins
(in order to call res_all$profiles_fivepr$five_prime_bins) or

10

get_metagene_data
P_sites_bins
(in order to call res_all$profiles_P_sites$P_sites_bins)
Read coverage for 50 bins between TSS and start codon, 100 bins for the CDS,
and 50 after stop codon to TES.
comp

String for originating compartment

Check for available originating compartments in the data set using: names(res_all$profiles_fivepr$f
profiles

5’ or P-site profile data generated by RiboseQC_analysis:
res_all$profiles_fivepr or
res_all$profiles_P_sites
Consists of DataFrames each containing counts of 5’ or P-site profiles, calculated for different resolution types (see parameter res), originating compartments (see parameter comp), and read lengths per input sample.
Example to access DataFrame:
res_all$profiles_fivepr[[res]][[comp]][[read_length]] or
res_all$profiles_P_sites[[res]][[comp]][[read_length]]

Value
This function returns data profile_data as list(data_single, data_all, res) with profile
data
• for all read lengths individually (profile_data[1]] is data_single) and
• for all read lenghts summarized (profile_data[[2]] is data_all).
with different scaling: no scaling (none), log2 scaling (log2), and z scoring (zscore), accessable
via e.g. profile_data[[1]]$none or profile_data[[2]]$zscore.

profile_data[[3]] saves the resolution type res for later use during plotting.

profile_data[[4]] stores information on whether data represents 5’ or P site profiles (profile_type)
for later use during plotting.
Author(s)
Dominique Sydow, 
See Also
create_html_report

get_ps_fromsplicemin

11

get_ps_fromsplicemin

Offset spliced reads on minus strand

Description
This function calculates P-sites positions for spliced reads on the minus strand
Usage
get_ps_fromsplicemin(x, cutoff)
Arguments
x
cutoff

a GAlignments object with a cigar string
number representing the offset value

Value
a GRanges object with offset reads
Author(s)
Lorenzo Calviello, 

get_ps_fromspliceplus Offset spliced reads on plus strand

Description
This function calculates P-sites positions for spliced reads on the plus strand
Usage
get_ps_fromspliceplus(x, cutoff)
Arguments
x
cutoff

a GAlignments object with a cigar string
number representing the offset value

Value
a GRanges object with offset reads
Author(s)
Lorenzo Calviello, 

12

get_top50_all_genes

get_rl_and_cutoffs

Get selected read lengths and cutoffs

Description
This function retrieves data on selected read lengths (per originating compartment), e.g. their cutoff
values, frame preference and codon gain.
This data is used in the Ribo-seQC report in section 4.2.2 (displayed as table).
Usage
get_rl_and_cutoffs(rdata_list)
Arguments
rdata_list

List of RiboseQC analysis RData objects generated by generate_rdata_list.

Value
This function returns data.
Author(s)
Dominique Sydow, 
See Also
create_html_report

get_top50_all_genes

Get top 50 abundant genes (all genes)

Description
This function retrieves data on the top 50 abundant genes.
This data is used in the Ribo-seQC report in section 6 (displayed as table).
Usage
get_top50_all_genes(rdata_list)
Arguments
rdata_list

List of RiboseQC analysis RData objects generated by generate_rdata_list.

get_top50_cds_genes

13

Value
This function returns data to be displayed as table in the html report.
Author(s)
Dominique Sydow, 
See Also
create_html_report

get_top50_cds_genes

Get top 50 abundant genes (CDS regions for protein coding genes)

Description
This function retrieves data on the top 50 abundant CDS regions for protein coding genes.
This data is used in the Ribo-seQC report in section 6 (displayed as table).
Usage
get_top50_cds_genes(rdata_list)
Arguments
rdata_list

List of RiboseQC analysis RData objects generated by generate_rdata_list.

Value
This function returns data to be displayed as table in the html report.
Author(s)
Dominique Sydow, 
See Also
create_html_report

14

load_annotation

get_top50_mapping

Get top 50 mapping positions

Description
This function retrieves data on the top 50 positions (nucleotide resolution) are listed where most
reads (their 5’ end) map to, revealing possibly contaminating sequences.
This data is used in the Ribo-seQC report in section 5 (displayed as table).
Usage
get_top50_mapping(rdata_list)
Arguments
rdata_list

List of RiboseQC analysis RData objects generated by generate_rdata_list.

Value
This function returns data to be displayed as table in the html report.
Author(s)
Dominique Sydow, 
See Also
create_html_report

load_annotation

Load genomic features and genome sequence

Description
This function loads the annotation created by the prepare_annotation_files function
Usage
load_annotation(path)
Arguments
path

Full path to the *Rannot R file in the annotation directory used in the prepare_annotation_files funct

plot_codon_usage_bulk

15

Value
introduces a GTF_annotation object and a genome_seq object in the parent environment
Author(s)
Lorenzo Calviello, 
See Also
prepare_annotation_files

plot_codon_usage_bulk Plot bulk codon usage bar plots

Description
This function plots the codon usage summed up over all positions* (bulk codon usage) as bar plot
for a specific data type, originating compartment, and read length, as well as based on a user-defined
genetic code.
* Information on positions included in analysis:
Based on P-site corrected reads, the codon usage within CDS regions for protein coding genes is
examplatory calculated
• for the first 11 codons of the CDS (referred to as start),
• for 11 codons from the middle of the CDS (referred to as middle), and
• for the last 11 codons of the CDS (referred to as stop).
Usage
plot_codon_usage_bulk(codon_usage_data, sample = "",
output_rds_path = "")
Arguments
sample

String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
codon_usage_bulk_data
List containing codon usage bulk data and meta data, generated by get_codon_usage_data.
Value
This function returns a plot that can be integrated in the html report and that can be saved as RDS
object file.

16

plot_codon_usage_bulk_rmd

Author(s)
Dominique Sydow, 
See Also
create_html_report

plot_codon_usage_bulk_rmd
Plot bulk codon usage bar plots within the RMarkdown document

Description
This function generates iteratively all bulk codon usage bar plots; iteration over originating compartments, read length, and data type (codon count, read count, codon-read count ratio).
These plots are displayed in the Ribo-seQC report in section 8.
Usage
plot_codon_usage_bulk_rmd(data, sample = "", output_rds_path = "")
Arguments
data

Object (list of lists) generated by RiboseQC_analysis: res_all.

sample

String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
Value
This function returns iteratively all bulk codon usage plots for the html report and saves the same
plots as RDS object file.
Author(s)
Dominique Sydow, 
See Also
create_html_report, get_metagene_data, plot_metagene_hm

plot_codon_usage_positional

17

plot_codon_usage_positional
Plot positional codon usage heatmap

Description
This function plots the codon usage per nucleotide position* (positional codon usage) as heatmap
for a specific data type, originating compartment, read length, and scaling method, as well as based
on a user-defined genetic code.
* Information on positions included in analysis:
Based on P-site corrected reads, the codon usage within CDS regions for protein coding genes is
examplatory calculated
• for the first 11 codons of the CDS (referred to as start),
• for 11 codons from the middle of the CDS (referred to as middle), and
• for the last 11 codons of the CDS (referred to as stop).
Usage
plot_codon_usage_positional(codon_usage_data, scal, sample = "",
output_rds_path = "")
Arguments
sample

String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
codon_usage_bulk_data
List containing codon usage bulk data and meta data, generated by get_codon_usage_data.
Value
This function returns a plot that can be integrated in the html report and that can be saved as RDS
object file.
Author(s)
Dominique Sydow, 
See Also
create_html_report

18

plot_codon_usage_positional_rmd

plot_codon_usage_positional_rmd
Plot positional codon usage heatmaps within the RMarkdown document

Description
This function generates iteratively all positional codon usage heatmaps; iteration over originating
compartments, read length, data type (codon count, read count, codon-read count ratio), and scaling
method (none, log2, zscale).
These plots are displayed in the Ribo-seQC report in section 7.
Usage
plot_codon_usage_positional_rmd(data, sample = "",
output_rds_path = "")
Arguments
data

Object (list of lists) generated by RiboseQC_analysis: res_all.

sample

String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
Value
This function returns iteratively all positional codon usage plots for the html report and saves the
same plots as RDS object file.
Author(s)
Dominique Sydow, 
See Also
create_html_report

plot_frame_dist_boxplot

19

plot_frame_dist_boxplot
Plot frame coverage of 5’-site profiles

Description
This function plots the frame coverage of 5’-site profiles, i.e. the fraction of reads (their 5’ end) in
frame 0, 1, and 2 (defined by start codon), as boxplots (per-frame distributions over all transcripts)
for individual read lengths as well as for all read lengths summarized.
Usage
plot_frame_dist_boxplot(analysis_frame_cutoff, comp, sample = "",
output_rds_path = "")
Arguments
analysis_frame_cutoff
Object containing statistics on frame coverage (per originating compartment and
read length) generated by RiboseQC_analysis.

comp

sample
output_rds_path

Example:
res_all$selection_cutoffs$analysis_frame_cutoff
String for originating compartment

Check for available originating compartments in the data set using: names(res_all$profiles_fivepr$f
String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.

Details
This plot is used in the Ribo-seQC report in section 4.2.1.
Value
This function returns a plot that can be integrated in the html report and that can be saved as RDS
object file.
Author(s)
Dominique Sydow, 
See Also
create_html_report

20

plot_frame_dist_boxplot_rmd

plot_frame_dist_boxplot_rmd
Plot frame coverage of 5’-site profiles within the RMarkdown document

Description
This function generates iteratively all frame coverage boxplots (iteration over originating compartments).
These plots are displayed in the Ribo-seQC report in section 4.2.1.
Usage
plot_frame_dist_boxplot_rmd(analysis_frame_cutoff, sample = "",
output_rds_path = "")
Arguments
analysis_frame_cutoff
Object containing statistics on frame coverage (per originating compartment and
read length) generated by RiboseQC_analysis.
Example:
res_all$selection_cutoffs$analysis_frame_cutoff
sample

String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
Value
This function integrates plots in the html report.
Author(s)
Dominique Sydow, 
See Also
create_html_report

plot_metagene_bar

21

plot_metagene_bar

Plot 5’/P-site profile per read length as barplot

Description
This function plots a 5’ or P-site profile as barplot (for a specific originating compartment, resolution type, and read length).
This plot is used in the Ribo-seQC report in section 4.1/4.3.
Usage
plot_metagene_bar(metagene_data, rl, sample = "", output_rds_path = "")
Arguments
sample

String; sample name (selected from the input names iven in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
data

Profile data for a resolution type, specific originating compartment, and read
length.
Example:
res_all$profiles_fivepr$five_prime_subcodon$nucl$'30'
or
res_all$profiles_P_sites$five_prime_subcodon$nucl$'30'
Shown in bold are fixed list names, remaining list names should be adapted
to the resolution type, specific originating compartment, and read length of interest.

Value
This function returns a plot that can be integrated in the html report and that can be saved as RDS
object file.
Author(s)
Dominique Sydow, 
See Also
create_html_report

22

plot_metagene_hm

plot_metagene_bar_rmd Plot 5’/P-site profile per read length as barplot in R Markdown

Description
This function plots a 5’ or P-site profile as barplot (for a specific originating compartment, resolution type, and read length).
This plot is used in the Ribo-seQC report in section 4.1/4.3.
Usage
plot_metagene_bar_rmd(metagene_data, sample = "", output_rds_path = "")
Arguments
metagene_data

...

sample

String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
Value
...
Author(s)
Dominique Sydow, 
See Also
create_html_report

plot_metagene_hm

Plot 5’/P-site profiles (for all read lengths) as heatmap

Description
This function plots a 5’ or P-site profiles for all read lengths as heatmap (for a specific originating
compartment, resolution type, and scaling method).
This plot is used in the Ribo-seQC report in section 4.1/4.3.

plot_metagene_hm_rmd

23

Usage
plot_metagene_hm(metagene_data, scal, sample = "",
output_rds_path = "")
Arguments
scal

Scaling method: no scaling (none), log2 scaling (log2), or z scoring (zscore).

sample

String; sample name (selected from the input names iven in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
data_profile

Profile data for a specific originating compartment and resolution type, generated using get_metagene_data.

Value
This function returns a plot that can be integrated in the html report and #’ that can be saved as RDS
object file.
Author(s)
Dominique Sydow, 
See Also
create_html_report

plot_metagene_hm_rmd

Plot 5’/P-site profiles as heatmaps within the RMarkdown document

Description
This function generates iteratively all heatmap plots (iteration over originating compartments, resolution types, and scaling methods).
These plots are displayed in the Ribo-seQC report in section 4.1/4.3.
Usage
plot_metagene_hm_rmd(data, profile_type, sample = "",
output_rds_path = "")

24

plot_read_biotype_dist_1

Arguments
sample

String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
profiles

5’ or P-site profile data generated by RiboseQC_analysis:
res_all$profiles_fivepr or
res_all$profiles_P_sites
Consists of DataFrames each containing counts of 5’ or P-site profiles, calculated for different resolution types (see parameter res), originating compartments (see parameter comp), and read lengths per input sample.
Example to access DataFrame:
res_all$profiles_fivepr[[res]][[comp]][[read_length]] or
res_all$profiles_P_sites[[res]][[comp]][[read_length]]

Value
This function returns iteratively all 5’ or P-site profile plots for the html report and saves the same
plots as RDS object file.
Author(s)
Dominique Sydow, 
See Also
create_html_report, get_metagene_data, plot_metagene_hm

plot_read_biotype_dist_1
Plot read location distribution by biotype (and originating compartment)

Description
This function plots the read location distribution by biotype (and originating compartment) for one
input sample.
This plot is used in the Ribo-seQC report in section 1.1.
Usage
plot_read_biotype_dist_1(pos, sample, output_rds_path = "")

plot_read_biotype_dist_2

25

Arguments
pos

res_all$read_stats$positions generated by RiboseQC_analysis.
data.frame containing the number of reads per biotype (rows) and originating compartment (columns).

sample

String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
Value
This function returns a plot that can be integrated in the html report and that can be saved as RDS
object file.
Author(s)
Dominique Sydow, 
See Also
create_html_report

plot_read_biotype_dist_2
Plot read location distribution by originating compartment (and biotype)

Description
This function plots the read location distribution by originating compartment (and biotype) for all
input samples.
This plot is used in the Ribo-seQC report in section 1.2.
Usage
plot_read_biotype_dist_2(rdata_list, output_rds_path = "")
Arguments
rdata_list
List of RiboseQC analysis RData objects generated by generate_rdata_list.
output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.

26

plot_read_biotype_dist_by_length

Value
This function returns a plot that can be integrated in the html report and that can be saved as RDS
object file.
Author(s)
Dominique Sydow, 
See Also
create_html_report

plot_read_biotype_dist_by_length
Plot read length and location distribution (distribution per read
length)

Description
This funtion plots the biotype distribution for each originating compartment as distribtion per read
length for one input sample (displayed as read count and as read count fraction).
This plot is used in the Ribo-seQC report in section 3.2.
Usage
plot_read_biotype_dist_by_length(reads_summary, sample,
output_rds_path = "")
Arguments
reads_summary

res_all$read_stats$reads_summary generated by RiboseQC_analysis
List of DataFrames: one DataFrame for each originating compartment, each
DataFrame contains read counts per biotype (rows) and read lengths (columns).

sample

String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
Value
This function returns a plot that can be integrated in the html report and that can be saved as RDS
object file.

plot_read_length_dist

27

Author(s)
Dominique Sydow, 
See Also
create_html_report

plot_read_length_dist Plot read length distribution

Description
This function plots the read length distribution per originating compartment for one input sample.
This plot is used in the Ribo-seQC report in section 2.
Usage
plot_read_length_dist(rld, sample, output_rds_path = "")
Arguments
rld

res_all$read_stats$rld generated by RiboseQC_analysis
data.frame containing the number of reads per originating compartment (rows)
and read lengths (columns).

sample

String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
Value
This function returns a plot that can be integrated in the html report and that can be saved as RDS
object file.
Author(s)
Dominique Sydow, 
See Also
create_html_report

28

plot_read_length_dist_by_biotype

plot_read_length_dist_by_biotype
Plot read length and location distribution (distribution per biotype)

Description
This funtion plots the read length distribution for each originating compartment as distribtion per
biotype for one input sample (displayed as read count and as read count fraction).
This plot is used in the Ribo-seQC report in section 3.1.
Usage
plot_read_length_dist_by_biotype(reads_summary, sample,
output_rds_path = "")
Arguments
reads_summary

res_all$read_stats$reads_summary generated by RiboseQC_analysis
List of DataFrames: one DataFrame for each originating compartment, each
DataFrame contains read counts per biotype (rows) and read lengths (columns).

sample

String; sample name (selected from the input names given in the input_sample_names
parameter of create_html_report).

output_rds_path
String; full path to output folder for RDS object files created by this function.
Defaults to NOT save RDS; to save RDS, provide path to destination folder.
Value
This function returns a plot that can be integrated in the html report and that can be saved as RDS
object file.
Author(s)
Dominique Sydow, 
See Also
create_html_report

prepare_annotation_files

29

prepare_annotation_files
Prepare comprehensive sets of annotated genomic features

Description
This function processes a gtf file and a twobit file (created using faToTwoBit from ucsc tools:
http://hgdownload.soe.ucsc.edu/admin/exe/ ) to create a comprehensive set of genomic regions of
interest in genomic and transcriptomic space (e.g. introns, UTRs, start/stop codons). In addition,
by linking genome sequence and annotation, it extracts additional info, such as gene and transcript
biotypes, genetic codes for different organelles, or chromosomes and transcripts lengths.
Usage
prepare_annotation_files(annotation_directory, twobit_file, gtf_file,
scientific_name = "Homo.sapiens", annotation_name = "genc25",
export_bed_tables_TxDb = T, forge_BSgenome = T, create_TxDb = T)
Arguments
annotation_directory
The target directory which will contain the output files
twobit_file

Full path to the genome file in twobit format

gtf_file
Full path to the annotation file in GTF format
scientific_name
A name to give to the organism studied; must be two words separated by a ".",
defaults to Homo.sapiens
annotation_name
A name to give to annotation used; defaults to genc25
export_bed_tables_TxDb
Export coordinates and info about different genomic regions in the annotation_directory?
It defaults to TRUE
forge_BSgenome Forge and install a BSgenome package? It defaults to TRUE
create_TxDb

Create a TxDb object and a *Rannot object? It defaults to TRUE

Details
This function uses the makeTxDbFromGFF function to create a TxDb object and extract genomic
regions and other info to a *Rannot R file; the mapToTranscripts and mapFromTranscripts functions are used to map features to genomic or transcript-level coordinates. GTF file mist contain
"exon" and "CDS" lines, where each line contains "transcript_id" and "gene_id" values. Additional
values such as "gene_biotype" or "gene_name" are also extracted. Regarding sequences, the twobit
file, together with input scientific and annotation names, is used to forge and install a BSgenome
package using the forgeBSgenomeDataPkg function.

30

prepare_annotation_files
The resulting GTF_annotation object (obtained after runnning load_annotation) contains:
txs: annotated transcript boundaries.
txs_gene: GRangesList including transcript grouped by gene.
seqinfo: indicating chromosomes and chromosome lengths.
start_stop_codons: the set of annotated start and stop codon, with respective transcript and
gene_ids. reprentative_mostcommon,reprentative_boundaries and reprentative_5len represent the
most common start/stop codon, the most upstream/downstream start/stop codons and the start/stop
codons residing on transcripts with the longest 5’UTRs
cds_txs: GRangesList including CDS grouped by transcript.
introns_txs: GRangesList including introns grouped by transcript.
cds_genes: GRangesList including CDS grouped by gene.
exons_txs: GRangesList including exons grouped by transcript.
exons_bins: the list of exonic bins with associated transcripts and genes.
junctions: the list of annotated splice junctions, with associated transcripts and genes.
genes: annotated genes coordinates.
threeutrs: collapsed set of 3’UTR regions, with correspinding gene_ids. This set does not overlap
CDS region.
fiveutrs: collapsed set of 5’UTR regions, with correspinding gene_ids. This set does not overlap
CDS region.
ncIsof: collapsed set of exonic regions of protein_coding genes, with correspinding gene_ids. This
set does not overlap CDS region.
ncRNAs: collapsed set of exonic regions of non_coding genes, with correspinding gene_ids. This
set does not overlap CDS region.
introns: collapsed set of intronic regions, with correspinding gene_ids. This set does not overlap
exonic region.
intergenicRegions: set of intergenic regions, defined as regions with no annotated genes on either strand.
trann: DataFrame object including (when available) the mapping between gene_id, gene_name,
gene_biotypes, transcript_id and transcript_biotypes.
cds_txs_coords: transcript-level coordinates of ORF boundaries, for each annotated coding transcript. Additional columns are the same as as for the start_stop_codons object.
genetic_codes: an object containing the list of genetic code ids used for each chromosome/organelle.
see GENETIC_CODE_TABLE for more info.
genome_package: the name of the forged BSgenome package. Loaded with load_annotation
function.
stop_in_gtf: stop codon, as defined in the annotation.

Value
a TxDb file and a *Rannot files are created in the specified annotation_directory. In addition, a
BSgenome object is forged, installed, and linked to the *Rannot object

Author(s)
Lorenzo Calviello, 

RiboseQC_analysis

31

See Also
load_annotation, forgeBSgenomeDataPkg, makeTxDbFromGFF.

RiboseQC_analysis

Perform a Ribo-seQC analysis

Description
This function loads annotation created by the prepare_annotation_files function, and analyzes a
BAM file.
Usage
RiboseQC_analysis(annotation_file, bam_files, read_subset = T,
readlength_choice_method = "max_coverage", chunk_size = 5000000L,
write_tmp_files = T, dest_names = NA, rescue_all_rls = FALSE,
fast_mode = T, create_report = T, sample_names = NA,
report_file = NA, extended_report = F, pdf_plots = T,
stranded = T, normalize_cov = T)
Arguments
annotation_file
Full path to the annotation file (*Rannot). Or, a vector with paths to one annotation file per bam file.
bam_files

character vector containing the full path to the bam files

read_subset

Select readlengths up to 99 percent of the reads, defaults to TRUE. Must be of
length 1 or same length as bam_files.
readlength_choice_method
Method used to subset relevant read lengths (see choose_readlengths function); defaults to "max_coverage". Must be of length 1 or same length as
bam_files.
chunk_size
write_tmp_files

dest_names

the number of alignments to read at each iteration, defaults to 5000000, increase
when more RAM is available. Must be between 10000 and 100000000
Should output all the results (in *results_RiboseQC_all)? Defaults to TRUE.
Must be of length 1 or same length as bam_files.
character vector containing the prefixes to use for the result output files. Defaults
to same as bam_files

rescue_all_rls Set cutoff of 12 for read lengths ignored because of insufficient coverage. Defaults to FALSE. Must be of length 1 or same length as bam_files.
fast_mode

Use only top 500 genes to build profiles? Defaults to TRUE. Must be of length 1
or same length as bam_files.

create_report

Create an html report showing the RiboseQC analysis results. Defaults to TRUE

32

RiboseQC_analysis
sample_names

character vector containing the names for each sample analyzed (for the html
report). Defaults to "sample1", "sample2" ...

report_file

desired filename for for the html report file. Defaults to the first entry of bam_files
followed by ".html"

extended_report
creates a large html report including codon occupancy for each read length. Defaults to FALSE
pdf_plots

creates a pdf file for each produced plot. Defaults to TRUE

stranded

are the analyzed libraries strand-specific? TRUE, FALSE or "inverse". Defaults
to TRUE

normalize_cov

export normalized (sum to 1 million) bedgraph files for coverage tracks? Defaults to TRUE

Details
This function loads different genomic regions created in the prepare_annotation_files step,
separating features on different recognized organelles. The bam files is then analyzed in chunks to
minimize RAM usage.
The complete list of analysis and output is as follows:
read_stats: contains:
read length distribution (rld) per organelle, positions containes mapping statistics on different
genomic regions, reads_pos1 contains 5’ end mapping positions for each read, separated by read
length. counts_cds_genes: contains read mapping statistics on CDS regions of protein coding
genes, including gene symbols, counts, RPKM and TPM values counts_all_genes: is a similar
object, but contains statistics on all annotated genes. reads_summary: reports mapping statistics on
different genomic regions and divided by read length and organelle.
profiles_fivepr contains:
five_prime_bins: a DataFrame object (one for each read length and compartment) with signal values over 50 5’UTR bins, 100 CDS bins and 50 3’UTR bins; one representative transcript
(reprentative_mostcommon) is selected for each gene. five_prime_subcodon containes a similar
structure, but for 25nt downstream the Transcription Start Site (TSS), 25nt upstream start codons,
33nt donwstream the start codon, 33nt in the middle of the ORF, 33nt upstream the stop codon, 25nt
downstream the stop codon, and 25nt upstream the Transcription End Site (TES).
selection_cutoffs contains:
results_choice: containing the calculated cutoffs and selected readlengths, together with data
about the different methods. results_cutoffs has statistics about calculated cutoffs, while analysis_frame_cutoff
has extensive statistics concerning cutoff calculations and read length selection, see calc_cutoffs_from_profiles
for more details.
P_sites_stats: contains the list of calculated P_sites, from all reads (P_sites_all), uniquely mapping reads (P_sites_all_uniq), or uniquely mapping reads with mismatches (P_sites_uniq_mm).
junctions contains stastics on read mapping on annotated splice junctions. coverage for entire
reads (no 5’ends or P_sites-transformed) on different strands and for all and uniquely mapping
reads are also calculated.

RiboseQC_analysis

33

profiles_P_sites contains:
P_sites_bins: profiles for each organelle and read length around binned transcript locations.
P_sites_subcodon: profiles for each organelle and read length around transcript start/ends and
ORF start/ends.
Codon_counts: codon occurrences in the first 11 codons, middle 11 codons, and last 11 codons for
each ORF.
P_sites_percodon: P_sites counts on each codon, separated by ORF positions as described above.
Values are separated by organelle and read length.
P_sites_percodon_ratio: ratio of P_sites_percodon/Codon_counts, as a measure of P_site occupancy on each codon, divided again by organelle and read length, for different ORF positions.
sequence_analysis: contains a DataFrame object with the 50top mapping location in the genome,
with the corresponding DNA sequence, number of reads mapping (also in percentage of total n of
reads), and genomic feature annotation.
summary_P_sites: contains a DataFrame object summarizing the P_sites calculation and read
length selection, including statistics on percentage of total reads used.
Value
the function saves a "results_RiboseQC_all" R file appended to the bam_files path including the
complete list of outputs described here. In addition, bedgraph files for coverage value and P_sites
position is appended to the bam_files path, including also a summary of P_sites selection statistics,
a smaller "results_RiboseQC" R file used for creating a dynamic html report, and a "for_SaTAnn"
R object that can be used in the SaTAnn pipeline.
Author(s)
Lorenzo Calviello, 
See Also
prepare_annotation_files, calc_cutoffs_from_profiles, choose_readlengths, create_html_report.

Index
create_html_report, 4, 6, 8–10, 12–14,
16–28, 33
create_pdfs_from_rds_objects, 5

∗Topic Ribo-seQC
calc_cutoffs_from_profiles, 2
choose_readlengths, 3
create_html_report, 4
create_pdfs_from_rds_objects, 5
generate_rdata_list, 6
get_codon_usage_data, 7
get_default_rl_selection, 8
get_metagene_data, 9
get_ps_fromsplicemin, 11
get_ps_fromspliceplus, 11
get_rl_and_cutoffs, 12
get_top50_all_genes, 12
get_top50_cds_genes, 13
get_top50_mapping, 14
load_annotation, 14
plot_codon_usage_bulk, 15
plot_codon_usage_bulk_rmd, 16
plot_codon_usage_positional, 17
plot_codon_usage_positional_rmd,
18
plot_frame_dist_boxplot, 19
plot_frame_dist_boxplot_rmd, 20
plot_metagene_bar, 21
plot_metagene_bar_rmd, 22
plot_metagene_hm, 22
plot_metagene_hm_rmd, 23
plot_read_biotype_dist_1, 24
plot_read_biotype_dist_2, 25
plot_read_biotype_dist_by_length,
26
plot_read_length_dist, 27
plot_read_length_dist_by_biotype,
28
prepare_annotation_files, 29
RiboseQC_analysis, 31

forgeBSgenomeDataPkg, 31
generate_rdata_list, 6
get_codon_usage_data, 5, 7
get_default_rl_selection, 5, 8
get_metagene_data, 5, 9, 16, 23, 24
get_ps_fromsplicemin, 11
get_ps_fromspliceplus, 11
get_rl_and_cutoffs, 5, 12
get_top50_all_genes, 5, 12
get_top50_cds_genes, 5, 13
get_top50_mapping, 5, 14
load_annotation, 14, 31
makeTxDbFromGFF, 31
plot_codon_usage_bulk, 5, 15
plot_codon_usage_bulk_rmd, 5, 16
plot_codon_usage_positional, 5, 17
plot_codon_usage_positional_rmd, 5, 18
plot_frame_dist_boxplot, 5, 19
plot_frame_dist_boxplot_rmd, 5, 20
plot_metagene_bar, 5, 21
plot_metagene_bar_rmd, 5, 22
plot_metagene_hm, 5, 16, 22, 24
plot_metagene_hm_rmd, 5, 23
plot_read_biotype_dist_1, 5, 24
plot_read_biotype_dist_2, 5, 25
plot_read_biotype_dist_by_length, 5, 26
plot_read_length_dist, 5, 27
plot_read_length_dist_by_biotype, 5, 28
prepare_annotation_files, 15, 29, 33
RiboseQC_analysis, 3, 31

calc_cutoffs_from_profiles, 2, 4, 33
choose_readlengths, 3, 33
34



---

Source Exif Data:

File Type                       : PDF
File Type Extension             : pdf
MIME Type                       : application/pdf
PDF Version                     : 1.5
Linearized                      : No
Page Count                      : 34
Page Mode                       : UseOutlines
Author                          : 
Title                           : 
Subject                         : 
Creator                         : LaTeX with hyperref package
Producer                        : pdfTeX-1.40.18
Create Date                     : 2019:05:31 16:10:58-07:00
Modify Date                     : 2019:05:31 16:10:58-07:00
Trapped                         : False
PTEX Fullbanner                 : This is pdfTeX, Version 3.14159265-2.6-1.40.18 (TeX Live 2017/Debian) kpathsea version 6.2.3

EXIF Metadata provided by EXIF.tools